Biosensors
A biosensor measures the physical changes that a biological recognition layer bound to a solid transducer undergoes when it interacts with a sample containing targeted molecules. It therefore uses the capacity of some biomolecules (receptors) to specifically bind to (recognize) complementary biomolecules (ligands). The most typical interactions are complementary nucleic acid hybridization and antibody/antigen binding. Biosensors are increasingly sought after in fundamental biological studies, health sciences research, drug discovery and clinical diagnosis1-3. Depending on the measured physical change, biosensors can be classified as optical, electrical and mechanical biosensors.
Optical biosensors can mainly be divided into label-based detection and label-free detection. The most commonly used label-based biosensors are based on fluorescence-based detection; target molecules or biorecognition molecules are labeled with fluorescent labels, such as dyes; the intensity of the fluorescence signal indicates the amount of targeted molecules. Meanwhile, fluorescence-based detection is extremely sensitive and requires laborious labeling methods which can also interfere with the function of the biomolecule. In contrast, in label-free detection, the targeted molecules are not labeled or altered and are detected in their natural forms. A significant part of label-free optical sensors measures the refractive index change close to the sensor surface by exciting an evanescent field which exponentially decays into the bulk solution with a characteristic length between tens to hundreds of nanometers4. The surface plasmon resonance (SPR) method and localized surface plasmon resonance (LSPR) methods are the most popular among label-free optical biosensors.
Electrochemical devices have traditionally received the most attention among electrical biosensors5-7. These devices usually couple enzymes that produce or consume electrons upon substrate recognition to a transducer consisting of electrodes. Many of these enzymes specifically catalyze the reactions of clinically important analytes such as glucose, lactate, cholesterol, amino acids, urate, pyruvate, glutamate, alcohol, hydroxybutyrate, to name a few. Nanotechnology advances are also providing nanoscale electrical biosensors based on semiconductor nanotubes and nanowires, in which electrochemical gating occurs from a change in the local surface potential due to target binding8-10.
The quartz crystal microbalance has become one of the most established techniques among mechanical biosensors11-13. These devices are based on quartz crystal resonators (such as those used in watches) which are piezoelectric and therefore allow directly measuring crystal deformation using electrical methods. In these devices, resonance frequency is measured and related to the change in mass induced by the analyte binding to the recognition layer immobilized on the crystalline surface. A subclass of mechanical biosensors is called nanomechanical biosensors which makes the best use of the nanoscale size of at least one of their dimensions14-20.
Among the existing methods, the most satisfactory biosensors in the biomedical field include end-point detection bioassays such as ELISA. ELISAs are essential tools in the biomedical field due to their good sensitivity, assay simplicity, reliability and high performance.
On the other hand, devices such as lateral flow tests are of the utmost importance given the short analysis time needed and they have been satisfactorily miniaturized and simplified to the point that the test can even be done at home. However, the lowest analyte concentration which they can detect is usually up to 0.1 μM, which is not good enough for detecting many targets of biological importance. In comparison, ELISA requires a longer analysis time (˜1 hour), but offers better concentration sensitivity (˜1 pM).
The biodetection technique which can combine excellent sensitivity and specificity with a short analysis time, together with miniaturization potential, is still under research. Particularly, there is a high demand for techniques which can be integrated in a point-of-care device with credible sensitivity, quantification capacity and good dynamic interval. No technique providing same has been demonstrated until now. The capacity of being integrated in a point-of-care (POC) device means that detection protocols must be simple, small volumes of sample must be used and they must not require complicated preparation and/or washing steps or complex chemicals for preparing the samples and/or detection devices. A low cost for complete analyses and a long storage life also are requirements for obtaining a commercially viable product.
Nanoparticle-based Biosensors
There are approaches in the prior art that have been successful in many, although not all, of the challenges mentioned for POC devices. Use of nanoparticles (NPs) has played a role in this success. Particularly, gold nanoparticles and other noble metal nanoparticles have been used in analyte detection. The localized surface plasmon resonance (LSPR) in gold NPs shifts when the surrounding dielectric constant changes, such that shifts in the LSPR spectral peak facilitated by biomolecule binding provide a method for analyte detection in clinical samples. Different detection approaches using this phenomenon at a nanoscale are reviewed in reference 21.
In a satisfactory approach called plasmonic ELISA, the localized plasmon resonance shift facilitated by gold nanoparticle aggregation is used for coloring the detection label of very low concentrations of an analyte of interest. In reference 22, both PSA and the p24 antigen of the HIV-1 capsid are detected at concentrations of only 1×10−18 g/ml. In this method, the biocatalytic cycle of an enzyme generates colored NP solutions due to the fact that aggregated NPs are formed when the hydrogen peroxide concentration decreases. Analyte binding promotes NP aggregation, which in turn gives a blue color to the solution. This color change is used as a detection signal which can even be tracked at a glance and thus provides a low cost detection approach.
In another relevant methodology, labeling NPs with various DNA sequences provides the multiplexing capacity that metal NPs alone would not have, since they lack a range of color labels for labeling each specific reaction. The so-called bio-barcode method has not only been used for DNA detection, but rather it has also been used satisfactorily for protein detection. The bio-barcode is based on magnetic microparticle probes with antibodies that bind specifically to a target of interest, for example, a clinically relevant protein such as a prostate-specific antigen (PSA) (see reference 23) and nanoparticle probes that are encoded with DNA that is unique to the target protein of interest and antibodies that can sandwich the target captured by the microparticle probes. Magnetic separation of the complexed probes and target, followed by dehybridization of the oligonucleotides on the nanoparticle probe surface allows determining the presence of the target protein by identifying the oligonucleotide sequence released from the nanoparticle probe. Due to the fact that the nanoparticle probe carries with it a large number of oligonucleotides for the protein binding event, there is substantial signal amplification and the target protein can be detected at low concentrations (30 attomolar concentration). Alternatively, a polymerase chain reaction (PCR) in the oligonucleotide barcodes can boost the sensitivity to 3 attomolar. Comparable clinically accepted conventional assays have sensitivity limits of 3 picomolar, six orders of magnitude less sensitive than that observed with this method23. A limitation of this technique is the analysis time required, up to 100 minutes, given the need to separate the complexed probes and target from the sample solution and then identify the DNA labels. Quantification is also possible with this method. One approach is to perform PCR and/or gel electrophoresis, but these are methods that are not suitable for point-of-care applications and exclude the rapid analysis described in reference 25.
An alternative detection method is based on the scattered light spectral change when at least two NPs are placed close to one another26,27. The color change is due to a shift in the surface plasmon resonance of Au nanoparticles when at least two NPs are placed close to one another. This produces a detectable color shift and a change in the collected light intensity which can be measured optically. NP probe complexes always comprise two or more nanoparticles bound to a specific target analyte; this has been referred to as a light scattering complex. This has the advantage that only NP aggregates containing the analyte are detected. Non-aggregated particles, including those not containing the target analyte, are not detected in this method. This allows detecting NP aggregates in the presence of a significant excess of non-aggregated particles. This method has demonstrated excellent sensitivity, better than 10 femtomoles of an oligonucleotide. Within this method, use of evanescent illumination by means of a supporting waveguide and scatter-based colorimetric detection has been demonstrated to be 4 orders of magnitude better than absorbance-based spot tests (patent document EP1639370).
One way to eliminate the need for PCR amplification while at the same time maintaining a good multiplexing capacity is to hybridize GNP scattering complexes on a solid support functionalized with known sequences in defined positions, as is done in fluorescent arrays. Subsequent scanometric detection of the scattered light serves as a biosensing signal and the multiplexing capacity is obtained through the predefined positions of the known immobilized sequences. A way to amplify this optical signal is usually needed. A method for amplifying the scattered light signal of NP labels is the silver reduction promoted by nanoparticles28 or colorimetric response by enzyme catalysis on optically coated silicon substrates29. This method is used for amplifying the optical signal and also allows quantifying the amount of analyte in the sample30.
Nanomechanical Resonators-based Biosensors
Nanomechanical resonators have demonstrated unprecedented limits of detection in the mass detection of atoms and molecules in vacuum. The mass limits of detection have been recently pushed down to the yoctogram range, i.e., the mass of a single proton can be measured. Two components are essential to achieve mass sensitivity: devices with nanoscale dimensions and high quality factors (1000-100000) that imply measurements in vacuum. However, biomolecule detection must ideally be carried out in aqueous solutions, the natural environment in which biological processes occur. Nanomechanical resonators in liquids show a very low quality factor (1-10) as a result of viscous damping. Furthermore, the liquid is entrained along with the nanomechanical resonator, increases its effective mass and thus reduces the sensitivity. Miniaturization of the devices to the nanoscale does not improve these limitations. More importantly, biological detection requires many repetitive measurements that can only be achieved with disposable and cost/effective devices that can be easily both handled and measured. These requirements are fulfilled by commercially available microcantilever arrays, but not by the nanoscale mechanical resonators of the state of the art that are still manufactured at a low rate by nanofabrication techniques and are highly irreproducible in the dimensions and mechanical response. Furthermore, measurement of the resonant frequency of these devices in liquid is scientifically and technically challenging. These limitations have limited the success of nanomechanical resonators as biological sensors.
Nanomechanical Resonators with Mass Labels
Nanomechanical resonators have used NPs for amplifying the signal; herein, greater mass binding provided by the labels increases sensor mechanical response. Herein, reduction in the resonance frequency is related to the added mass of the analyte-NP complex which binds to the resonator. Despite the fact that dynamic nanomechanical sensors have demonstrated a good performance without labels, labeling greatly improves specificity and can reduce the limit of detection. It has been demonstrated that sample labeling for nanomechanical detection is advantageous in end-point assays. Craighead et al. demonstrated in reference 31 that labeling a monoclonal antibody with nanoparticles in a sandwich-type immunoassay improved the limit of detection by three orders of magnitude to reach 2 ng/ml in prion protein detection and to even detect the presence of 50 fg/ml of enriched PSA in a background noise of fetal bovine serum. The technique is also quantitative since the authors found a clear linear dependence of the frequency response on PSA concentration. The capacity for detecting fM concentrations of a target protein in a realistic background noise places labeled resonant cantilever sensors in an excellent position for competing with the other innovative techniques mentioned, in addition to more established technologies. Nevertheless, nanomechanical resonators are still not widely used in clinical practice. That is due to the fact that they lack the necessary robustness in response. The few studies showing a statistically significant number of tests show that the number of false positives and false negatives is still too high. The frequency shift commonly used as detection signal in these sensors depends largely on the non-specific adsorption on the device surface.
A key limitation of nanomechanical resonators is non-specific adsorption. The final limits of detection predicted by theoretical approaches can be far from the actual limits of detection when nanomechanical biosensors functionalized with bioreceptors are immersed in complex solutions, such as serum, for detecting the presence of biomarkers in real time or ex-situ. In this situation, other molecules at a much higher concentration, even trillions of times higher, are present in the solution. Although these molecules have lesser affinity for the receptors grafted with sensors, their high concentration confers the actual limit of detection. For example, cancer biomarkers are in blood plasma at a concentration in the range of 1 ng/ml, whereas the concentration of undesired proteins is about 70 mg/ml. Most nanomechanical biosensors meet the sensitivity for achieving cancer biomarker detection. However, the selectivity determining the false positive and false negative rates has received little attention. Cancer marker detection in complex media such as serum requires selectivity greater than 1 part per million.
Theoretical predictions indicate that the selectivity required for biomarker detection in complex media can be achieved by functionalizing the sensors with a high surface receptor density32. This prediction is according to the findings in surface tension-based nanomechanical biosensors, in which the best results are obtained at high receptor packing densities. A second theoretical prediction is that the step of additional intermediate surface passivation by small inert molecules after receptor incubation could significantly reduce biofouling and aid in achieving better selectivity. Interestingly, the size and geometry of the blocking molecule used to refill the empty spaces on the sensor surface plays a critical function. That is according to the recent statistical analysis results of the effect of immunoreactions on nanomechanical biosensor response in the static mode33. The study comprised 1012 cantilevers with different antibody surface densities, two blocking strategies based on polyethylene glycol (PEG) and bovine serum albumin (BSA), meticulous controls with non-specific antibodies and small proteins such as lysozymes. The study showed that the performance of the assay depends critically on both the antibody surface density and the blocking strategies. It was found that optimal conditions involve antibody surface densities near but below saturation and blocking with PEG.
Furthermore, other practical approaches for minimizing non-specific adsorption and enhancing selectivity has been proposed. Use of arrays of nanomechanical elements with an internal reference aids in rejecting common noise sources, including non-specific adsorption. Another approach is the implementation of the sandwich assays conventionally used in ELISA. In this assay, the nanomechanical system is functionalized with a molecular receptor specific for the biomarker of interest. After exposure of the nanomechanical system to the sample, the device is incubated with secondary receptors bound to a molecule or a material acting as a signal amplifier, such as a nanoparticle, for increasing the mass effect. Use of two different receptors greatly enhances sensitivity and specificity. This approach was applied for detecting prion proteins with nanomechanical resonator, which in conformationally altered forms are known to cause neurodegenerative diseases in animals as well as human beings34. The resonance frequency was detected ex situ in high vacuum. For the direct incubation of the nanomechanical resonators functionalized with a primary antibody against the prion protein, the limit of detection was about 20 μg/ml. When the resonators were subjected to a subsequent step of incubation with bound secondary antibodies, the limit of detection was enhanced 3 orders of magnitude, being about 2 ng/ml.
A second promising strategy that maintains the natural label-free characteristic of nanomechanical biosensors is to implement microfluidics for sample purification and preconcentration. The potential of this approach has been demonstrated with label-free nanowire nanosensors. In this work, a microfluidic purification chip simultaneously captures multiple biomarkers from blood samples and releases them, after washing, into purified buffer for detection by nanosensors8. This two-stage approach isolates the detector from the complex environment of whole blood, and reduces its minimum required sensitivity by effectively pre-concentrating the biomarkers. The authors demonstrated quantitative and specific detection of two model cancer antigens from a 10 ml sample of whole blood in less than 20 min.
Although nanotechnology has provided biosensors with unpredictable sensitivity levels without the need of labeling, nanosensors have also shown significant difficulties in issues relating to specificity and reproducibility, and they are therefore still not ready for biomarker selection in blood. This arises from the extreme difficulty in ‘finding’ low-abundance protein biomarkers in a ‘haystack’ of plasma proteins, some of them at concentrations at least seven orders of magnitude higher (albumin about 40 mg/ml). Therefore, the situation is that the high biological noise perceived by non-specific interactions greatly exceeds the intrinsic noise of most existing nanosensors. In short, the problem is not sensitivity, but rather:                Specificity, for discriminating traces of biomarkers in the complex blood protein mixture.        Reliability, for minimizing distressing false positives and false negatives in patient diagnosis.        
The authors of the present invention have now found a system for biodetection applications which allows ultra-low limits of detection since it discriminates concentrations around 10 ag/ml. Furthermore, the system allows target analyte detection in complex biological background noises, such as for example, blood samples, without needing any purification step. The invention is based on a sandwich-type optical assay which takes advantage of the surprising and unexpected enhancement of the plasmonic effect caused in the nanoparticles by the combination of the particular nature and design of the substrate used in the biosensor and the particular nature and dimensions of the nanoparticle. This system can be adapted in a nanomechanical device for the purpose of analyzing both optoplasmonic and mechanical signals such that it improves detection reliability. The robustness of this dual biosensor leads to extremely low false positive and false negative rates, ≈2×10−4 at an ultra-low concentration of 100 ag/ml, thereby providing an excellent solution for being integrated in a POC device.